The sulfhydryl amino acids are metabolized according to a complex set of pathways as shown in FIG. 1.
As can be seen, methylation of homocysteine to form methionine via methionine synthetase requires methylcobalamin (Me-Cbl), also known as methyl-B.sub.12. The methyl group is donated by N.sup.5 -methyltetrahydrofolate (N.sup.5 -MTHF), which is converted to tetrahydrofolate (THF). Conversion of methylmalonyl-CoA to succinyl-CoA via methylmalonyl-CoA mutase requires adenosyl-cobalamin, also known as adenosyl-B.sub.12 as a cofactor. Thus cobalamin and folic acid are vital cofactors in sulfhydryl metabolism and cobalamin, but not folic acid, is a vital cofactor in methylmalonyl-CoA metabolism.
Accurate and early diagnosis of cobalamin and folate deficiencies in warm-blooded animals is important because these deficiencies can lead to life-threatening hematologic abnormalities which are completely reversible by treatment with cobalamin or folate, respectively. Accurate and early diagnosis of cobalamin deficiency is especially important because it can also lead to incapacitating and life-threatening neuropsychiatric abnormalities; administration of exogenous cobalamin always stops the progression of these abnormalities, almost always leads to significant improvement in symptoms, and frequently leads to their complete correction. The distinction between cobalamin and folate deficiency is often difficult because both deficiencies lead to indistinguishable hematologic abnormalities; the distinction is important because use of the proper vitamin results in the greatest improvement in hematologic abnormalities and, more importantly, only cobalamin will correct the neuropsychiatric abnormalities which are only seen in cobalamin deficiencies. The use of folic acid to treat cobalamin deficiency is extremely dangerous, since some, or all, of the hematolcgic abnormalities may improve, but neuropsychiatric abnormalities will not improve and may progress, or even be precipitated.
Chapters in leading medical textbooks and articles in leading medical journals teach that cobalamin deficiency should be suspected in individuals with significant anemia (i.e. decreased hematocrit or hemoglobin) in whom the red blood cells are macrocytic (i.e., mean cell volume (MCV)generally&gt;100 fl), or in individuals who have neurologic abnormalities consisting of peripheral neuropathy and/or ataxia. Many such textbook chapters or journal articles further state that the anemia is typically severe, i.e. hemoglobin .ltoreq.8 g %, hematocrit &lt;25%, and size of the red blood cells is greatly increased to levels &gt;110 fl. [See, e.g., Babior, B. M., and H. F. Bunn, in Harrison's Principles of Internal Medicine (R. G. Petersdorf, R. F. Adams, E. Braunwald, K. J. Isselbacher, J. B. Martin, and J. D. Wilson, eds.)(McGraw-Hill Book Co., New York, 1983), pp. 1853-1860; Lee, G. R., and H. J. Gardner, in Textbook of Family Practice, 3rd Ed. (R. E. Rakel, ed.)(W. B. Saunders & Co., Philadelphia, 1984), pp. 1082-1091.]
Several laboratory tests have been reported as giving abnormal results in patients with cobalamin deficiency or folate deficiency. Such tests include measurements of red blood cell folate [Hoffbrand, A. V., et al., J. Clin. Path. 19:17(1966)]and the "dU suppression test"[Metz, J., et al., Brit. J. Haem. 14:575 (1968)], but neither is widely utilized. It has been known for more than twenty years that methylmalonic acid is excreted in increased amounts in the urine of most patients with cobalamin deficiency and that this abnormality is evidenced by only a few patients with folate deficiency. Because it is believed and taught that cobalamin deficiency should be suspected and can be diagnosed accurately based on the presence and degree of anemia, the presence and degree of macrocytosis, and by the presence and degree of depressed serum cobalamin levels, methylmalonic acid is rarely measured in patients suspected of being cobalamin-deficient. Indeed, it is taught in a leading textbook of medicine and in a leading textbook of hematology that in practice, assay of urinary methylmalonate is rarely necessary. [Beck, W. S. , in Hematology, 3rd Ed. (W. J. Williams, E. Beutler, A. J. Erslev, and M. A. Lichtman, eds.) (McGraw-Hill Book Co., New York, 1983), pp. 434-465; Beck, W. S. , in Cecil Textbook of Medicine, Vol. 1 (J. B. Wyngaarden and L. H. Smith, Jr., eds)(W. B. Saunders Co., Philadelphia, 1985), pp. 893-900.] While one recent journal article does advocate measurement of urinary methylmalonic acid [Norman, E. J., O. J. Martelo, and M. D. Denton, Blood 59(6):1128-1131 (1982)], analysis of the data in this paper reveals that 26 of the 27 cobalamin-deficient patients were anemic, that 23 of 27 patients had an elevated MCV, and that 12 of 12 patients whose serum cobalamin was measured with the standard improved serum cobalamin assay had values below 100 pg/ml. Thus these patients were all cobalamin-deficient according to standard diagnostic procedures and additional assays would normally have been judged to be unnecessary.
Assays for cobalamin and folate in serum or plasma are the most widely utilized and recommended tests for diagnosing and distinguishing cobalamin and folate deficiency. In the case of cobalamin deficiency, in which the normal range for serum cobalamin is about 200-900 pg/ml, several leading authors state that patients will not only have low serum cobalamin levels, but that these values will be below 100 pg/ml. [See, e.g., Babior, supra; Lee & Gardner, supra; Beck, in Textbook of Medicine, supra; and Beck, in Hematology, supra].
In 1978, it was discovered that cobalamin analogues are present in human plasma and that their presence could mask cobalamin deficiency because the radioisotope dilution assays for serum cobalamin then in use were not specific for true cobalamin. This problem could be corrected by using pure or purified intrinsic factor as the binding protein in the radioisotope dilution assay for cobalamin and this modification has almost totally replaced assays existing in 1978 that used a nonspecific cobalamin-binding protein. See, e.g., U.S. Pat. No. 4,188,189 (Allen), U.S. Pat. No. 4,351,822 (Allen), U.S. Pat. No. 4,451,571 (Allen), and Kolhouse, J. F., H. Kondo, N. C. Allen, E. Podell, and R. H. Allen, N. Eng. J. Med. 299:785-792 (1978). These improved assays for serum cobalamin are now utilized in thousands of laboratories throughout the world and appear to give low values for all, or almost all, patients with cobalamin deficiency.
The improved assays have been severely criticized, however, because they frequently give low values in patients who lack any evidence of cobalamin deficiency. Because of this, experts in the field have taught that cobalamin deficiency should be considered, and serum cobalamin values should be obtained, only in patients who have hematologic or neurologic abnormalities that are typical of patients with cobalamin deficiency, as discussed above. Schilling and his coworkers, experts in the field of cobalamin deficiency and laboratory diagnosis, have stated:
"We conclude that the `improved` vitamin B.sub.12 assay kits will yield an increased proportion of clinically unexplained low results for serum B.sub.12. PA0 It seems prudent for scientific and economic reasons to measure serum vitamin B.sub.12 only in patients who have hematological or neurological findings that suggest a reasonable probability of vitamin B.sub.12 deficiency. Measuring serum B.sub.12 as a screening test in the anemic or the geriatric population will result in a high proportion of low values that cannot be correlated with clinical disease."
Schilling, R. F., V. F. Fairbanks, R. Miller, K. Schmitt, and M. J. Smith, Clin. Chem. 29(3):582-583 (1983). Thus, the presently available widely used cobalamin assays may frequently provide low serum cobalamin levels in patients who are not truly cobalamin deficient. Such findings are confusing or misleading to the physician and may result in unneccessary and expensive further testing.
Thus, it is generally taught in the art that the clinical spectrum of cobalamin deficiency is relatively narrow and well-defined and that the possibility of cobalamin deficiency should only be considered in those who have concurrent hematological or neurological symptoms, i.e., usually those patients with moderately severe anemia accompanied by moderately severe macrocytosis, and in those patients with peripheral neuropathy and/or ataxia. Routine screening of the general population or those with only moderate anemia, or moderate macrocytosis, or other neuropsychiatric abnormalities, would lead to high numbers of false positives.
It has now been discovered that the clinical spectrum of cobalamin deficiency is much broader than previously recognized and that many cobalamin-deficient patients are not anemic, or only moderately anemic; that in many cases their red blood cells are not macrocytic, or only moderately macrocytic; that in many cases a variety of neurologic abnormalities other than peripheral neuropathy and ataxia are present; and that in many cases the serum cobalamin level is only slightly decreased and may actually be normal, even with the improved assays above using purified intrinsic factor. Accordingly, there is a need for an improved assay for cobalamin deficiency, preferably one in which cobalamin deficiency can be distinguished from folate deficiency.